Detection of Rotavirus from diarrhoeic cow calves in Mathura , India

Diarrhoeal diseases are important cause of morbidity and mortality in neonates of various animal species. Rotaviruses cause neonatal diarrhea in calves. The present study was carried out to determine the prevalence of rotavirus infection among cow calves in Mathura and adjacent regions. During the present study 101 diarrheic and 29 non diarrheic stool samples collected from cow calves were screened for rotavirus. Of the101 diarrheic samples 17 samples (16.83%) were found to be positive for rotavirus by RNA PAGE. All the non-diarrheic samples were negative for rotavirus. All the isolates exhibited 4-2-3-2 migration pattern suggesting group A rotavirus. Depending on migration of 10th and 11th segments, all the isolates were of long pattern. Three different electropherotypes were detected in this study period. Male diarrheic calves were found to be more susceptible to rotavirus infection (20.37%) than female diarrheic calves (12.76%). Besides Rotavirus antigen was detected by ELISA.


Introduction
diarrheic and 29 non diarrheic) were collected from both organized and non-organized dairy farms located Rotaviruses belong to family Reoviridae.They in and around Mathura during study period 2007pose as major etiologic agents of acute gastroenteritis 2009.The stool samples were collected in sterilized in the various mammalian species including humans plastic container, transported under ice and stored atand calves [8].Rotaviruses, are non-enveloped, o 20 C till further processing.icosahedral particles consisting of 11 segments of Preparation of fecal suspension: 10% fecal double-stranded RNA (dsRNA) enclosed in a triplesuspension was prepared in phosphate buffer saline layered protein capsid [2].Rotaviruses are classified (PBS, pH7.2).It was centrifuged at 10,000 rpm for 30 in to seven groups: A to G and several subgroups based minutes.1ml of this fecal supernatant was used for o on specificity of VP6 inner shell polypeptide.RNA extraction and rest was stored at -70 C.However, the strains of rotaviruses are further Extraction of viral RNA from fecal supernatant: classified into electropherotypes on the basis of Viral RNA extraction was done using phenol:chloroform differences in the relative migration rates of genome method as described by Herring et al. [6].The pellet segments in PAGE, creating more opportunities for was suspended in 2X RNA sample buffer for RNA strain diversification [9].Analysis of electrophoretic PAGE analysis and stored at -20°C until required.mobilities of the segments of dsRNA by PAGE yields Screening by RNA-PAGE: Electrophoresis of a pattern, which is constant, and characteristic for a extracted RNA was performed as per the method particular rotavirus isolate [4].Conventional described by Laemmli [12] and Herring et al. [6] in techniques like electron microscopy (EM), isolation in 7.5% resolving and 5% stacking gel.The silver MA-104 cell line, electropherotyping, and various staining of the gel was done as described by Herring et serological tests are used for diagnosis of rotavirus al. [6].The stained gel was photographed and stored in infection [3].In this paper we have used RNA PAGE 10% ethanol (Fig. 1).as a standard technique along with ELISA.
Screening by ELISA: All the stool samples were again screened for the presence of rotavirus antigen by pattern (Zone I, II, III and IV) suggesting group A Any sample that yielded a difference of 0.15 or rotavirus [14].The results showed that 11 of greater in optical density was considered positive 54(20.37%)male calves were found positive whereas (Table -3).

Results and Discussion
susceptibility of male bovine calves (42.85%) in In this study we selected electropherotyping comparison to female calves (28.2%).In contrast to (RNA-PAGE) as the primary identifier of rotavirus our findings Hasso and Pandey [5] observed that strains in feces of diarrheic calves due to the following female calves were more prone to rotavirus infection.reasons.Each rotavirus strain reveals a single distinct Age wise susceptibility was also evaluated.The electro-pherotype upon PAGE.Analysis of the results indicated that newborn calves of first 8 weeks electrophoretic mobility of the 11 segments of ds RNA of age were more susceptible to rotavirus infection by PAGE yields a pattern which is both constant and (Table -2).All the 17 rotavirus positive samples were characteristic for a particular rotavirus isolate [4].from diarrheic calves under the age of 8 weeks.Similar Besides, it is easier to collect fecal samples with results were recorded by Sharma [15] in bovine calves.respect to serum samples.All samples positive for Electrophoretic pattern of the rotavirus positive Materials and methodsELISA.ELISA was performed to detect rotavirusCollection of specimens: 130 fecal samples (101 antigen in the fecal supernatants as described by the kit manufacturer (Rotavirus ELISA kit, Bio-X rotavirus were subjected to PAGE thrice to confirm the Diagnostics).The 96 well plate provided by the kit reproducibility of the migration pattern of the genome.In the present study we have analyzed fecal samples contains two different capture antibodies.Rows A, C, obtained from single diarrheic episode.Out of 101 E, G were coated with rotavirus specific capture fecal samples processed from diarrheic calves 17 antibodies and rows B, D, F, H coated with non samples were found positive for rotavirus by RNA specific antibodies.The detection antibody present in PAGE.The overall prevalence was 16.83% in the kit is is a peroxidase labelled antirotavirus specific diarrheic calves.However, in other studies rotavirus monoclonal antibody.The net optical density of each prevalence of 45.11, 34.5 and 4.3% have been reported sample was calculated by subtracting the reading for from different parts of the country [7, 10 and 13].None each sample well from corresponding negative control. of the non-diarrheic sample was positive.All the RNA Net optical density (O.D.) = O.D. of specific binding -O.D. of PAGE positive samples exhibited 4-2-3-2 migration non-specific binding.

Figure- 1 .
Figure-1.Electrophoretic migration patterns of different rotavirus isolates belonged to group A & displayed a long genome pattern.Lane1: SA11 reference strain.

Table - 3. Net absorbance values representing samples positive for rotavirus by ELISA
b4 to B51 are samples from calves